Members of the genus Rickettsia, including Rickettsia prowazekii, the etiological agent of epidemic typhus, are unique, even amongst the obligate intracellular bacterial parasites. These organisms grow directly in the cytoplasm, rather than within a vacuole in the cytoplasm of their eucaryotic host cells. We remain committed to understanding the biochemical and physiological mechanism by which R. prowazekii copes with the problems and exploits the opportunities of this unique environmental niche. This proposal has overall four areas of research focus. 1. Phospholipase and penetration: The putative components of rickettsial penetration (entry and exit) will be investigated: a) The substrate specificity and enzyme source (rickettsial or host) of the phospholipase will be characterized using liposomes which contain either rickettsial antibodies as a quasi-receptor or phospholipids labeled in specified acyl groups (as targets for the rickettsiae or suitable for fusion with host cells). b) The elucidation of induced phagocytosis as monitored by actin polymerization will be correlated with the other facets of induced phagocytosis. c) Intracellular damage to organelles by rickettsiae-dependent phospholipase will be investigated. d) The role of phospholipase in the exit of the rickettsiae will be defined though the use of specific phospholipase inhibitors and chloramphenicol. Appropriate phospholipase inhibitors may allow the rickettsiae to grow and divide in the originally infected cells but not allow their spread to adjacent cells. e) Extracorporeal rickettsial antigens will be characterized and their role in rickettsial infection in general and with the phospholipase specifically will be established. 2. Host Cell Mutants: We will select, isolate and characterize mutant CHO cells that do not support the normal entry or growth of R. prowazekii and therefore survive as uninfected cells in long term culture in the presence of rickettsiae. 3. Cell Wall: a) The metabolism of UDPG to insoluble polysaccharide in isolated rickettsiae and in a cell-free system will be characterized. b) The formation of diaminopimelic acid, the role of lysine in this formation, and the incorporation of these compounds into the rickettsial murein will be examined in rickettsiae whilst they are growing in their host cells, i.e., in situ. 4. Nucleotide Transport and Metabolism: a) The ATP/ADP Translocase molecule, from both rickettsiae and E. coli clones expressing the rickettsial translocase gene, will be purified and characterized employing reconstitution into proteoliposomes. b) The RNA Polymerase of R. prowazekii will be characterized with respect to its subunit composition, its interaction with promoters on the ricketettsial DNA, and its association with the rickettsial membrane or ribosomes. c) The Ribonucleotide Reductase of R. prowazekii will be characterized both in vitro and in situ with respect to its regulatory properties and as an alternative to the transport of DNXPs in which NXPs are transported and then reduced to dNXP. d) The Thymidylate Synthetase of R. prowazekii will be investigated in situ as the means for rickettsiae obtaining dTXP in lieu of transport and in concert with ribonucleotide reductase. e) The transport and synthesis of rickettsial Polyamines, putative nucleic acid stabilizers and metabolic activators, will be characterized.